Cannabinoid Production Research Articles

The bioengineering of cannabis

Genetic modification could enable industrial-scale production of cannabinoids that have pharmaceutical potential. Genetic modification could enable industrial-scale production of cannabinoids that have pharmaceutical potential.

Demetrix pursues THC, CBD from yeast

Demetrix has raised $50 million in a first round of venture funding to establish high-volume fermentation production of tetrahydrocannabinol (THC), cannabidiol (CBD), and other cannabinoids. Backers include Tuatara Capital and Horizons Ventures. The California-based start-up was cofounded in 2015 by University of California, Berkeley, chemical engineering professor Jay Keasling. Demetrix says it will use a modified strain of baker’s yeast, Saccharomyces cerevisiae, to produce the cannabinoids. The firm is targeting drug, supplement, and consumer product markets.

Signaling compounds elicit expression of key genes in cannabinoid pathway and related metabolites in cannabis

The potential for product recovery of metabolites can be improved by several methods; one of the most common techniques is elicitation. Elicitors can adjust multiple control points and precipitate the expression of some key genes. This project was conducted with the aim of increasing valuable secondary metabolites such as D-9-tetrahydrocannabinol (THC) and cannabidiol (CBD) identified in cannabis plants. Whole plants were treated with salicylic acid (SA) and γ-Aminobutyric acid (GABA) to evaluate their effect on THCAS, CBDAS, OLS, and PT genes which are responsible for production of the main cannabinoids. THC and CBD contents in related samples were measured using HPLC, 72 h after treatments. THCAS expression was enhanced and reached its maximum level with 1 mM salicylic acid; however, the expression of CBDAS, OLS, and PT decreased. Furthermore, treatment with GABA enhanced the THCAS expression mainly at 0.1 mM. The highest THC content was obtained in 1 mM salicylic acid and 0.1 mM GABA treatments, respectively. The results suggested that salicylic acid and GABA can control the signaling cascades of genes in cannabinoid pathway by changing their expression patterns at critical concentration, and these two compounds can be considered as effective elicitors for commercial cannabinoid production. Designing an appropriate standard protocol based on spraying approach on plants to elicit specific metabolites such as THC and CBD which are mainly produced at flowering time and in flowering organs has many benefits alongside other elicitation approaches.

Complete biosynthesis of cannabinoids and their unnatural analogues in yeast.

Cannabis sativa L. has been cultivated and used around the globe for its medicinal properties for millennia1. Some cannabinoids, the hallmark constituents of Cannabis, and their analogues have been investigated extensively for their potential medical applications2. Certain cannabinoid formulations have been approved as prescription drugs in several countries for the treatment of a range of human ailments3. However, the study and medicinal use of cannabinoids has been hampered by the legal scheduling of Cannabis, the low in planta abundances of nearly all of the dozens of known cannabinoids4, and their structural complexity, which limits bulk chemical synthesis. Here we report the complete biosynthesis of the major cannabinoids cannabigerolic acid, Δ9-tetrahydrocannabinolic acid, cannabidiolic acid, Δ9-tetrahydrocannabivarinic acid and cannabidivarinic acid in Saccharomyces cerevisiae, from the simple sugar galactose. To accomplish this, we engineered the native mevalonate pathway to provide a high flux of geranyl pyrophosphate and introduced a heterologous, multi-organism-derived hexanoyl-CoA biosynthetic pathway5. We also introduced the Cannabis genes that encode the enzymes involved in the biosynthesis of olivetolic acid6, as well as the gene for a previously undiscovered enzyme with geranylpyrophosphate:olivetolate geranyltransferase activity and the genes for corresponding cannabinoid synthases7,8. Furthermore, we established a biosynthetic approach that harnessed the promiscuity of several pathway genes to produce cannabinoid analogues. Feeding different fatty acids to our engineered strains yielded cannabinoid analogues with modifications in the part of the molecule that is known to alter receptor binding affinity and potency9. We also demonstrated that our biological system could be complemented by simple synthetic chemistry to further expand the accessible chemical space. Our work presents a platform for the production of natural and unnatural cannabinoids that will allow for more rigorous study of these compounds and could be used in the development of treatments for a variety of human health problems.

A cell-free platform for the prenylation of natural products and application to cannabinoid production

Prenylation of natural compounds adds structural diversity, alters biological activity, and enhances therapeutic potential. Because prenylated compounds often have a low natural abundance, alternative production methods are needed. Metabolic engineering enables natural product biosynthesis from inexpensive biomass, but is limited by the complexity of secondary metabolite pathways, intermediate and product toxicities, and substrate accessibility. Alternatively, enzyme catalyzed prenyl transfer provides excellent regio- and stereo-specificity, but requires expensive isoprenyl pyrophosphate substrates. Here we develop a flexible cell-free enzymatic prenylating system that generates isoprenyl pyrophosphate substrates from glucose to prenylate an array of natural products. The system provides an efficient route to cannabinoid precursors cannabigerolic acid (CBGA) and cannabigerovarinic acid (CBGVA) at >1 g/L, and a single enzymatic step converts the precursors into cannabidiolic acid (CBDA) and cannabidivarinic acid (CBDVA). Cell-free methods may provide a powerful alternative to metabolic engineering for chemicals that are hard to produce in living organisms.

Elucidation of structure-function relationship of THCA and CBDA synthase from Cannabis sativaL.

Cannabinoids are secondary natural products from the plant Cannabis sativaL. Therapeutic indications of cannabinoids currently comprise a significant area of medicinal research. We have expressed the Δ9-tetrahydrocannabinolic acid synthase (THCAS) and cannabidiolic acid synthase (CBDAS) recombinantly in Komagataella phaffii and could detect eight different products with a cannabinoid scaffold after conversion of the precursor cannabigerolic acid (CBGA). Besides five products remaining to be identified, both enzymes were forming three major cannabinoids of C. sativa - Δ9-tetrahydrocannabinolic acid (THCA), cannabidiolic acid (CBDA) and cannabichromenic acid (CBCA). In pursuit of improved enzyme properties for a biotechnological cannabinoid production, we performed site-directed mutagenesis to investigate the glycosylation pattern, the C-terminal berberine-bridge-enzyme (BBE) domain, the active site and the product specificity of both enzymes. The THCAS variant T_N89Q+N499Q (lacking two glycosylation sites) exerted about two-fold increased activity compared to wild-type enzyme. Variant T_H494C+R532C (additional disulfide bridge) exerted about 1.7-fold increased activity compared to wild-type enzyme and a shifted temperature optimum from 52 °C to 57 °C. We generated two CBDAS variants, C_S116A and C_A414V, with 2.8 and 3.3-fold increased catalytic activities for CBDA production. C_A414V additionally showed a broadened pH spectrum and a 19-fold increased catalytic activity for THCA production. These studies lay the groundwork for further research as well as biotechnological cannabinoid production.

The Effect of Light Spectrum on the Morphology and Cannabinoid Content of Cannabis sativa L.

Cannabis sativa L. flowers are the main source of Δ-9-tetrahydrocannabinol (THC) used in medicine. One of the most important growth factors in cannabis cultivation is light; light quality, light intensity, and photoperiod play a big role in a successful growth protocol. The aim of the present study was to examine the effect of 3 different light sources on morphology and cannabinoid production. Cannabis clones were grown under 3 different light spectra, namely high-pressure sodium (HPS), AP673L (LED), and NS1 (LED). Light intensity was set to ∼450 µmol/m²/s measured from the canopy top. The photoperiod was 18L: 6D/21 days during the vegetative phase and 12L: 12D/46 days during the generative phase, respectively. At the end of the experiment, plant dry weight partition, plant height, and cannabinoid content (THC, cannabidiol [CBD], tetrahydrocannabivarin [THCV], cannabigerol [CBG]) were measured under different light treatments. The experiment was repeated twice. The 3 light treatments (HPS, NS1, AP673L) resulted in differences in cannabis plant morphology and in cannabinoid content, but not in total yield of cannabinoids. Plants under HPS treatment were taller and had more flower dry weight than those under treatments AP673L and NS1. Treatment NS1 had the highest CBG content. Treatments NS1 and AP673L had higher CBD and THC concentrations than the HPS treatment. Results were similar between experiments 1 and 2. Our results show that the plant morphology can be manipulated with the light spectrum. Furthermore, it is possible to affect the accumulation of different cannabinoids to increase the potential of medicinal grade cannabis. In conclusion, an optimized light spectrum improves the value and quality of cannabis. Current LED technology showed significant differences in growth habit and cannabinoid profile compared to the traditional HPS light source. Finally, no difference of flowering time was observed under different R:FR (i.e., the ratio between red and far-red light).

Activation of polyketide synthase gene promoter in Cannabis sativa by heterologous transcription factors derived from Humulus lupulus

Cannabis sativa, an annual herbaceous plant, produce wide variety of secondary metabolites among which delta-9-tetrahydrocannabinol (THC) is the most important one. The dissection of biosynthetic pathway(s) of this compound and its regulation by transcription factors (TFs) is an important prerequisite for efficient biotechnological manipulation of its secondary metabolome. A polyketide synthase (PKS) of C. sativa catalyzes the first step of cannabinoid biosynthesis, leading to the biosynthesis of olivetolic acid. Cloning and analysis of PKS promoter based on online PLACE, PlantCARE, and Genomatix Matinspector professional databases, indicated that PKS promoter consisted of cis-elements such as TATA-box, CAAT-box, W-box, Myb-box, E-box, and P-box. Plant expression vector PKS::GUS was constructed in such a way that the ATG of the PKS gene was in the frame with the β-glucuronidase (GUS) coding region. Using a combinatorial transient GUS expression system in Nicotiana benthamania leaves, it was shown that heterologous TFs such as HlWRKY1, HlMYB3, HlWDR1 and HlbZIP1 from Humulus lupulus significantly activated PKS promoter. Moreover, Tombusvirus p19 core protein, which is known for silencing suppressor functions, acted in our combinatorial transient expression system as an enhancer of PKS promoter activity along with hop TFs. Our analyses suggested the involvement of the hop derived TFs (HlWRKY1, HlMYB3, HlWDR1 and HlbZIP1A) and p19 in the activation of PKS gene promoter, which could be used for the genetic manipulation of C. sativa to enhance the cannabinoid production.

Current Perspectives on Biotechnological Cannabinoid Production in Plants.

The plant Cannabis sativa contains a number of psychoactive chemical compounds, the cannabinoids, which possess a significant pharmaceutical potential. Recently, the usage of Cannabis for medicinal purposes was legalized in many countries. Thus, the study on the influence of different cannabinoids in combination with other Cannabis-derived compounds with respect to the treatment of various diseases becomes increasingly important. Besides the production of distinct cannabinoids in a heterologous host, like tobacco or yeast, transgenic Cannabis plants would be a suitable alternative to modify and therefore optimize the cannabinoid profile. This perspective highlights the current efforts on Cannabis cell culture systems, in vitro propagation, and transformation of the plant and reveals the resulting opportunities concerning biotechnological production of cannabinoids. Furthermore, alternative platform organisms for the heterologous production of cannabinoids, like tobacco, are considered and evaluated.

The application of plant in vitro cultures in cannabinoid production

Cannabinoids have considerable interest in the pharmaceutical industry. However, the production of medicines from hemp (Cannabis sativa L.) in most countries is restricted by law. Large-scale, field cultivation of hemp is difficult to control. Cannabinoid content in plants is variable and depends on multiple factors. Therefore, alternative methods of production have been investigated. The development of micropropagation techniques is a necessary step for genetic modification. Promising results have been obtained for certain narcotic genotypes. However, micropropagation of fibre types requires further research. Hemp can be genetically modified which may contribute to the breeding of new varieties in the future. Cell suspension cultures and hairy root cultures of hemp have been used to produce cannabinoids but obtaining cannabinoids from callus and cell suspension cultures has proved impossible. Adventitious roots can, however, deliver small amounts of these metabolites but production ceases over time and is too low for industrial applications.

Designing microorganisms for heterologous biosynthesis of cannabinoids.

During the last decade, the use of medical Cannabis has expanded globally and legislation is getting more liberal in many countries, facilitating the research on cannabinoids. The unique interaction of cannabinoids with the human endocannabinoid system makes these compounds an interesting target to be studied as therapeutic agents for the treatment of several medical conditions. However, currently there are important limitations in the study, production and use of cannabinoids as pharmaceutical drugs. Besides the main constituent tetrahydrocannabinolic acid, the structurally related compound cannabidiol is of high interest as drug candidate. From the more than 100 known cannabinoids reported, most can only be extracted in very low amounts and their pharmacological profile has not been determined. Today, cannabinoids are isolated from the strictly regulated Cannabis plant, and the supply of compounds with sufficient quality is a major problem. Biotechnological production could be an attractive alternative mode of production. Herein, we explore the potential use of synthetic biology as an alternative strategy for synthesis of cannabinoids in heterologous hosts. We summarize the current knowledge surrounding cannabinoids biosynthesis and present a comprehensive description of the key steps of the genuine and artificial pathway, systems biotechnology needs and platform optimization.

Δ9-Tetrahydrocannabinolic acid synthase: The application of a plant secondary metabolite enzyme in biocatalytic chemical synthesis

Δ9-Tetrahydrocannabinolic acid synthase (THCAS) from the secondary metabolism of Cannabis sativa L. catalyzes the oxidative formation of an intramolecular CC bond in cannabigerolic acid (CBGA) to synthesize Δ9-tetrahydrocannabinolic acid (THCA), which is the direct precursor of Δ9-tetrahydrocannabinol (Δ9-THC). Aiming on a biotechnological production of cannabinoids, we investigated the potential of the heterologously produced plant oxidase in a cell-free system on preparative scale. THCAS was characterized in an aqueous/organic two-liquid phase setup in order to solubilize the hydrophobic substrate and to allow in situ product removal. Compared to the single phase aqueous setup the specific activity decreased by a factor of approximately 2 pointing to a substrate limitation of CBGA in the two-liquid phase system. However, the specific activity remained stable for at least 3h illustrating the benefit of the two-liquid phase setup. In a repeated-batch setup, THCAS showed only a minor loss of specific activity in the third batch pointing to a high intrinsic stability and high solvent tolerance of the enzyme. Maximal space-time-yields of 0.121gL−1h−1 were reached proving the two-liquid phase concept suitable for biotechnological production of cannabinoids.

Cannabinoids provide the runner's reward

Neuroscience The “runner's high”—beneficial effects of prolonged exercise that reduce anxiety and pain perception—in mice appears to depend on the production of endogenous cannabinoids rather than endorphins. Mice allowed to do their normal running on a wheel (about 5 km per day) had increased circulating concentrations of β-endorphin (an opioid) and anandamide (an endocannabinoid). Fuss et al. found that the depletion of cannabinoid receptor 1 in neurons of the forebrain reduced the beneficial effects of running on anxiety-like behavior and tolerance to a painful stimulus. Proc. Natl. Acad. Sci. U.S.A. 10.1073/pnas.1514996112 (2015).

Hydroxylation and glycosylation of Δ9-tetrahydrocannabinol by Catharanthus roseus cell suspension culture

Δ9-tetrahydrocannabinol is the active constituent in Cannabis sativa, with reported analgesic, anti-emetic, anti-oxidative, neuroprotective, and anti-inflammatory activities. Δ9-THC has been used to treat a number of disease states including pain, anxiety, asthma, glaucoma, and hypertension. Poor water solubility of Δ9-THC greatly reduces its clinical effectiveness. Consequently, there is a need to modify the compound to increase its polarity and pharmaceutical efficacy. The aim of this study was to test the capability of Catharanthus roseus suspension cultured cells to convert Δ9-THC into more polar derivatives. The transformed metabolites were analyzed and isolated by HPLC. Structures of some new derivatives were proposed on the basis of molecular ion peaks and fragmentation patterns obtained from LC-MS and UV spectra obtained by HPLC, respectively. Δ9-THC was rapidly absorbed by Catharanthus roseus cultured cells and upon biotransformation new glycosylated and hydroxylated derivatives were isolated by preparative HPLC. In addition, cannabinol was detected as degradation product, including its glycosylated derivative. Based on these results, it is concluded that Catharanthus cultured cells have great potential to transform Δ9-THC into more polar derivatives and can be used for the large scale production of new cannabinoids, which can be a source of new compounds with interesting pharmacological profiles.

Cannabis micropropagation – Applications and updates

Cannabis sativa L. is one of the oldest plants yielding fiber, food and medicine. This species is a natural source of Δ9-Tetrahydrocannabinol (Δ9-THC) that has a tremendous therapeutic value and therefore, of high commercial interest in the pharmaceutical market. Vegetative propagation under indoor growing facilities is currently being used for conservation and maintenance of the genetic integrity of the elite clones. However, multiplication by this method is limited based on the availability and growth of screened mother plants. Micropropagation of cannabis using tissue culture techniques can be employed as an alternative to the conventional method of propagation and can play a vital role in the rapid multiplication, development of novel germplasm and genetic modification for enhanced cannabinoid production. Recent research on in vitro propagation of C. sativa has resulted in the development of protocols through direct organogenesis, indirect organogenesis and synthetic seed technology. This report provides an overview of recent developments, their current applications and the challenges that remain in micropropagation of C. sativa.

Therapeutic Potential of Cannabinoids—Perspectives for the Future

Isolation of cannabinoids from Cannabis and chemical production of synthetic cannabinoids resulted in understanding of their mechanism of action. Presently, renewed interest in therapeutic potential of cannabinoids is observed. There are numerous studies in the range of chemistry, pharmacology, and molecular biology that investigate the possibilities of their therapeutic application. This review presents results of research on cannabinoids with an emphasis on THC and its effect on a progression of selected diseases. The paper discusses previous reports and outlines future directions.

Cannflavins from hemp sprouts, a novel cannabinoid-free hemp food product, target microsomal prostaglandin E2 synthase-1 and 5-lipoxygenase

Hemp seeds are of great nutritional value, containing all essential amino acids and fatty acids in sufficient amount and ratio to meet the dietary human demand. Hemp seeds do not contain cannabinoids, and because of their high contents of ω-3 fatty acids, are enjoying a growing popularity as a super-food to beneficially affect chronic inflammation. Seeds also lack the typical phenolics of hemp leaves and inflorescences, but we found that sprouting, while not triggering the production of cannabinoids, could nevertheless induce the production of the anti-inflammatory prenylflavonoids cannflavins A and B. This effect was especially marked in Ermo, a cannabinoid-free variety of Cannabis sativa L. Microsomal prostaglandin E2 synthase (mPGES-1) and 5-lipoxygenase (5-LO) were identified as the molecular targets of cannflavins A and B, solving an almost three-decade old uncertainty on the mechanism of their the anti-inflammatory activity. No change on the fatty acid profile was observed during sprouting, and the presence of lipophilic flavonoids combines with the high concentration of ω-3 essential acids to qualify sprouts from Ermo as a novel anti-inflammatory hemp food product worth considering for mass production and commercial development.

Analysis of cannabinoids in laser-microdissected trichomes of medicinal Cannabis sativa using LCMS and cryogenic NMR

Trichomes, especially the capitate-stalked glandular hairs, are well known as the main sites of cannabinoid and essential oil production of Cannabis sativa. In this study the distribution and density of various types of Cannabis sativa L. trichomes, have been investigated by scanning electron microscopy (SEM). Furthermore, glandular trichomes were isolated over the flowering period (8 weeks) by laser microdissection (LMD) and the cannabinoid profile analyzed by LCMS. Cannabinoids were detected in extracts of 25-143 collected cells of capitate-sessile and capitate stalked trichomes and separately in the gland (head) and the stem of the latter. Δ(9)-Tetrahydrocannabinolic acid [THCA (1)], cannabidiolic acid [CBDA (2)], and cannabigerolic acid [CBGA (3)] were identified as most-abundant compounds in all analyzed samples while their decarboxylated derivatives, Δ(9)-tetrahydrocannabinol [THC (4)], cannabidiol [CBD (5)], and cannabigerol [CBG (6)], co-detected in all samples, were present at significantly lower levels. Cannabichromene [CBC (8)] along with cannabinol (CBN (9)) were identified as minor compounds only in the samples of intact capitate-stalked trichomes and their heads harvested from 8-week old plants. Cryogenic nuclear magnetic resonance spectroscopy (NMR) was used to confirm the occurrence of major cannabinoids, THCA (1) and CBDA (2), in capitate-stalked and capitate-sessile trichomes. Cryogenic NMR enabled the additional identification of cannabichromenic acid [CBCA (7)] in the dissected trichomes, which was not possible by LCMS as standard was not available. The hereby documented detection of metabolites in the stems of capitate-stalked trichomes indicates a complex biosynthesis and localization over the trichome cells forming the glandular secretion unit.

Opposite effects of methanandamide on lipopolysaccharide-induced prostaglandin E2 and F2α synthesis in uterine explants from pregnant mice.

Prostaglandins (PG) are effective abortifacients and are important mediators of lipopolisaccharide (LPS)-induced embryonic resorption (ER). Besides, anandamide (AEA) has been described as one of the major endocannabinoids present in the uterus suggesting that it might play a role in reproduction. It has been reported that high levels of AEA are associated with pregnancy failure and that LPS increases AEA production. Also, it has been observed that AEA modulates PG production in different tissues. In this sense, we studied whether LPS-induced PG production is modulated by AEA and we also assessed the effect of this endocannabinoid on PG metabolism in an in vitro model. Uterine explants from BALB/c implantation sites were cultured in the presence of LPS plus cannabinoid receptor (CB) specific antagonists and PG production was assessed. Then, we studied the effect of exogenous AEA on different steps of PG metabolic pathway. We showed that AEA is involved in LPS-induced PG biosynthesis. Also, we observed that AEA exerts opposite effects on PGE2 and PGF2α biosynthesis, by inhibiting PGE2 production and increasing PGF2α levels. We suggest that AEA could be involved in the mechanisms implicated in LPS-induced ER. A better understanding of how AEA could be affecting ER could help developing specific interventions to prevent this pathology.

Endogenous Cannabinoid Production in the Rat Female Reproductive Tract Is Regulated by Changes in the Hormonal Milieu

The endogenous cannabinoid (eCB) system is emerging as an important component of female reproductive tract physiology. The eCBs anandamide (AEA), 2-arachidonoyl glycerol (2-AG), and N-arachidonoyl glycine (NAGly) were measured in the rat reproductive tract at five time points in the four-day estrous cycle, in acyclic retired breeders (RB), after ovariectomy (OVX), OVX + estrogen (E2), OVX + progesterone (P4), or OVX with E2+P4. eCBs were measured in the uterus, uterine adipose, ovaries, and ovarian adipose using HPLC/MS/MS. Levels of AEA, 2-AG, and NAGly were highest in the estrus phase of the estrous cycle in the uterus, whereas, only NAGly had differences in production in the ovaries across the cycle. All eCBs were lower in RB ovaries; however, the production of eCBs in the uterus of RB and OVX groups was more varied with NAGly showing the lowest levels of production in these groups. Levels of AEA in uterine fat were significantly higher or equivalent to levels in the uterus. However, levels of 2-AG and NAGly were dramatically lower in uterine fat verses the organ. Ovarian fat had significantly lower levels of all three eCBs. These data provide evidence that the hormonal milieu plays a significant and complex role in the production of eCBs in the female rat reproductive tract.